Methods of seed breeding using high throughput nondestructive seed sampling

ABSTRACT

The present invention provides for novel methods to facilitate germplasm improvement activities through the use of high throughput, nondestructive sampling of seeds. A method of evaluating seeds for a desired trait, for example, for facilitating germplasm improvement activities, etc., generally includes removing tissue samples from individual seeds using an automated seed sampler without affecting germination viability of the seeds, analyzing the tissue samples for one or more characteristics indicative of a desired trait, and selecting the sampled seeds that include the desired trait.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/108,762 filed May 16, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/767,640 filed Apr. 26, 2010 (now U.S. Pat. No.7,941,969 issued May 17, 2011), which is a continuation of U.S. patentapplication Ser. No. 11/680,611 filed Feb. 28, 2007 (now U.S. Pat. No.7,703,238 issued Apr. 27, 2010), which claims priority to U.S.Provisional Application Ser. No. 60/778,828 filed Mar. 2, 2006, andwhich is a continuation-in-part of U.S. patent application Ser. No.11/213,435 filed Aug. 26, 2005 (now U.S. Pat. No. 7,611,842 issued Nov.3, 2009). U.S. patent application Ser. No. 11/213,435 claims priority toU.S. Provisional Application Ser. No. 60/604,604 filed Aug. 26, 2004,and U.S. Provisional Application Ser. No. 60/691,100 filed Jun. 15,2005. The entire disclosures of all of the above applications areincorporated herein by reference.

FIELD

The present invention relates to the field of plant breeding. Morespecifically, this invention provides methods for augmenting andeconomizing germplasm improvement activities using high throughput andnondestructive seed sampling techniques.

BACKGROUND

The statements in this section merely provide background informationrelated to the present disclosure and may not constitute prior art.

In plant development and improvement, genetic improvements are made inthe plant, either through selective breeding or genetic manipulation,and when a desirable improvement is achieved, a commercial quantity isdeveloped by planting and harvesting seeds over several generations. Tospeed up the process of plant improvement, statistical samples are takenand tested to advance seeds from the population that have inherited orexhibit the desired trait. However this statistical sampling necessarilyallows some seeds without the desirable trait to remain in thepopulation, and also can inadvertently exclude some seeds with thedesirable trait from the desired population. Not all seeds inherit orexhibit the desired traits, and thus these seeds still need to be culledfrom the population.

Apparatus and methods for the high-throughput, non-destructive samplingof seeds have been described which would overcome the obstacles ofstatistical samples by allowing for individual seed analysis. Forexample, U.S. patent application Ser. No. 11/213,430 (filed Aug. 26,2005); U.S. patent application Ser. No. 11/213,431 (filed Aug. 26,2005); U.S. patent application Ser. No. 11/213,432 (filed Aug. 26,2005); U.S. patent application Ser. No. 11/213,434 (filed Aug. 26,2005); and U.S. patent application Ser. No. 11/213,435 (filed Aug. 26,2005), which are incorporated herein by reference in their entirety,disclose apparatus and systems for the automated sampling of seeds aswell as methods of sampling, testing and bulking seeds.

The present invention addresses needs in the art for improved breedingmethods using high-throughput, non-destructive seed sampling systems.

SUMMARY

The present disclosure relates to systems and methods for facilitatinggermplasm improvement activities through the use of high throughput,nondestructive sampling of seeds. With automated, non-destructivesampling, it is possible to test individual seeds in a population, andselect only the seeds that possess one or more desired characteristics.This allows for new and more efficient methods for germplasm improvementand management, which lead to improved breeding populations.

In one embodiment, the present disclosure provides for ahigh-throughput, non-destructive method for analyzing individual seedsin a population of seeds. The method comprises removing a sample from aplurality of seeds in the population while preserving the germinationviability of the seed and analyzing the sample for the presence orabsence of one or more characteristics indicative of at least onegenetic or chemical trait.

In a further embodiment, the present disclosure provides for ahigh-throughput method for analyzing a population of haploid seed. Themethod comprises removing a sample from a plurality of seeds in apopulation of haploid seed while preserving the germination viability ofthe seed and analyzing the samples for the presence or absence of one ormore characteristics indicative of at least one genetic or chemicaltrait.

In a still further embodiment, the present disclosure provides for ahigh-throughput method for bulking a population of doubled haploid seed.The method comprises providing a population of seeds comprising haploidseeds and selecting one or more individual seeds exhibiting at least onepreferred characteristic from the population of seeds. Doubled haploidseeds are then produced from the selected seeds and a sample is removedfrom each doubled haploid seed while preserving the germinationviability of the seeds. The samples are analyzed for the presence orabsence of one or more characteristics indicative of at least onegenetic or chemical trait. Based on the results of the analysis, one ormore individual doubled haploid seeds are selected and plants or planttissue is cultivated from the selected doubled haploid seed.

In the various embodiments of the present invention, the samples may beanalyzed for one or more characteristics indicative of at least onechemical trait. Examples of such characteristics may include proteins,oils, carbohydrates, fatty acids, amino acids, biopolymers,pharmaceuticals, starch, fermentable starch, secondary compounds, andmetabolites.

In other various embodiments of the present invention, the samples maybe analyzed for one or more characteristics indicative of at least onegenetic trait. Examples of such characteristics may include a geneticmarker, a single nucleotide polymorphism, a simple sequence repeat, arestriction fragment length polymorphism, a haplotype, a tag SNP, analleles of a genetic marker, a gene, a DNA-derived sequence, anRNA-derived sequence, a promoter, a 5′ untranslated region of a gene, a3′ untranslated region of a gene, microRNA, siRNA, a QTL, a satellitemarker, a transgene, mRNA, ds mRNA, a transcriptional profile, and amethylation pattern.

Further areas of applicability of the present teachings will becomeapparent from the description provided herein. It should be understoodthat the description and specific examples are intended for purposes ofillustration only and are not intended to limit the scope of the presentteachings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings described herein are for illustration purposes only and arenot intended to limit the scope of the present disclosure in any way.

FIG. 1 is an allelogram depicting maize endosperm tissue samples thathave undergone PCR for detection of a particular SNP as described inExample 3.

FIG. 2 is a graphical illustration of the efficacy of pre-selection ondriving the frequency of favorable haplotypes as described in Example 6.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is notintended to limit the present disclosure, application, or uses.

The present invention provides for novel methods to facilitate germplasmimprovement activities through the use of high throughput,nondestructive sampling of seeds. The methods are useful in analyzingseeds in order to identify and select seeds comprising one or moredesired traits, markers, and genotypes. In one aspect of the invention,the analytical methods allow individual seeds that are present in abatch or a bulk population of seeds to be analyzed such that thechemical and/or genetic characteristics of the individual seeds can bedetermined.

Samples prepared by the present invention can be used for determining awide variety of physical, morphological, chemical and/or genetic traits.Generally, such traits are determined by analyzing the samples for oneor more characteristics indicative of at least one genetic or chemicaltrait. Non-limiting examples of characteristics indicative of chemicaltraits include proteins, oils, carbohydrates, fatty acids, amino acids,biopolymers, pharmaceuticals, starch, fermentable starch, secondarycompounds, and metabolites. Accordingly, non-limiting examples ofchemical traits include amino acid content, protein content, starchcontent, fermentation yield, fermentation efficiency, energy yield, oilcontent, determination of protein profiles determination of fatty acidprofiles, determination of metabolite profiles, etc.

Non-limiting examples of characteristics indicative of genetic traitsmay include, for example, genetic markers, single nucleotidepolymorphisms, simple sequence repeats, restriction fragment lengthpolymorphisms, haplotypes, tag SNPs, alleles of genetic markers, genes,DNA-derived sequences, RNA-derived sequences, promoters, 5′ untranslatedregions of genes, 3′ untranslated regions of genes, microRNA, siRNA,quantitative trait loci (QTL), satellite markers, transgenes, mRNA, dsmRNA, transcriptional profiles, and methylation patterns.

In one embodiment, the sampling of endosperm tissue enables thedetermination of allele frequencies, whereby it is possible to inferparental linkage phase for a particular marker. Further, comparison ofallele frequency data between two or more germplasm pools providesinsight into the targets of selection, whereby alleles increasing infrequency in conjunction with a shift in distribution of one or moretraits are presumed to be linked to said trait or traits of interest.Also, evaluation of relative allele frequency data between lines cancontribute to the construction of genetic linkage maps.

In another embodiment, the methods of the present invention use highthroughput, nondestructive seed sampling with doubled haploidtechnologies to contribute to germplasm improvement activities includingeconomization of doubled haploid programs by selecting only preferredseed for doubling, high throughput analysis of haploid and doubledhaploid material for both genotypic and chemical characteristics, traitintegration and evaluation, and marker-assisted breeding.

The methods and devices of the present invention can be used in abreeding program to select plants or seeds having a desired genetic orchemical trait, wherein a desired genetic trait comprises a genotype, ahaplotype, an allele, a sequence, a transcript profile, and amethylation pattern. The methods of the present invention can be used incombination with any breeding methodology and can be used to select asingle generation or to select multiple generations. The choice ofbreeding method depends on the mode of plant reproduction, theheritability of the trait(s) being improved, and the type of cultivarused commercially (e.g., F₁ hybrid cultivar, pureline cultivar, etc).Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. It is further understood that anycommercial and non-commercial cultivars can be utilized in a breedingprogram. Factors including, for example, without limitation, emergencevigor, vegetative vigor, stress tolerance, disease resistance,branching, flowering, seed set, seed size, seed density, standability,and threshability will generally dictate the choice.

In a particular embodiment, the methods of the present invention areused to determine the genetic characteristics of seeds in amarker-assisted breeding program. Such methods allow for improvedmarker-assisted breeding programs wherein nondestructive direct seedsampling can be conducted while maintaining the identity of individualseeds from the seed sampler to the field. As a result, themarker-assisted breeding program results in a “high-throughput” and moreefficient platform wherein a population of seeds having a desired trait,marker or genotype can be more effectively bulked in a shorter period oftime, with less field and labor resources required. Such advantages willbe more fully described below.

In one embodiment, the present invention provides a method for analyzingindividual seeds within a population of seeds having geneticdifferences. The method comprises removing a sample comprising cellswith nucleic acids from seeds in the population without affecting thegermination viability of the seeds; analyzing the nucleic acidsextracted from the sample for the presence or absence of at least onegenetic marker; selecting seeds from the population based upon theresults of the nucleic acid analysis; and cultivating plants from theselected seed.

As described above, the sampling systems and methods of this inventionprotect germination viability of the seeds so as to be non-destructive.Germination viability means that a predominant number of sampled seeds,(i.e., greater than 50% of all sampled seeds) remain viable aftersampling. In a particular embodiment, at least about 75% of sampledseeds, and in some embodiments at least about 85% of sampled seedsremain viable. It should be noted that lower rates of germinationviability may be tolerable under certain circumstances or for certainapplications, for example, as genotyping costs decrease with timebecause a greater number of seeds could be sampled for the same genotypecost. It should also be noted that sampling does not need to have anyeffect on viability at all.

In another embodiment, germination viability is maintained for at leastabout six months after sampling to ensure that the sampled seed will beviable until it reaches the field for planting. In a particularembodiment, the methods of the present invention further comprisetreating the sampled seeds to maintain germination viability. Suchtreatment may generally include any means known in the art forprotecting a seed from environmental conditions while in storage ortransport. For example, in one embodiment, the sampled seeds may betreated with a polymer and/or a fungicide to protect the sampled seedwhile in storage or in transport to the field before planting.

In one embodiment, the samples of the present invention are used in ahigh-throughput, non-destructive method for analyzing individual seedsin a population of seeds. The method comprises removing a sample fromthe seed while preserving the germination viability of the seed; andanalyzing the sample for the presence or absence of one or morecharacteristics indicative of a genetic or chemical trait. The methodmay further comprise selecting seeds from the population based on theresults of the analysis; and cultivating plants or plant tissue from theselected seed.

DNA may be extracted from the sample using any DNA extraction methodsknown to those of skill in the art which will provide sufficient DNAyield, DNA quality, PCR response, and sequencing methods response. Anon-limiting example of suitable DNA-extraction methods is SDS-basedextraction with centrifugation. In addition, the extracted DNA may beamplified after extraction using any amplification method known to thoseskilled in the art. For example, one suitable amplification method isthe GenomiPhi® DNA amplification prep from Amersham Biosciences.

Further, RNA may be extracted from the sample using any RNA extractionmethods known to those of skill in the art which will provide sufficientRNA yield, RNA quality, PCR response, and sequencing methods response. Anon-limiting example of suitable RNA-extraction methods is SDS-basedextraction with centrifugation with consideration for RNase-freereagents and supplies. In addition, the extracted RNA may be amplifiedafter extraction using any amplification method known to those skilledin the art. For example, one suitable amplification method is the FullSpectrum™ RNA Amplification from System Biosciences.

The extracted nucleic acids are analyzed for the presence or absence ofa suitable genetic polymorphism. A wide variety of genetic markers forthe analysis of genetic polymorphisms are available and known to thoseof skill in the art. As used herein, genetic markers include, but arenot limited to, simple sequence repeats (SSRs), single nucleotidepolymorphisms (SNPs), insertions or deletions (Indels), single featurepolymorphisms (SFPs, for example, as described in Borevitz et al. 2003Gen. Res. 13:513-523) or transcriptional profiles, and nucleic acidsequences. A nucleic acid analysis for the presence or absence of thegenetic marker can be used for the selection of seeds in a breedingpopulation. The analysis may be used to select for genes, QTL, alleles,or genomic regions (haplotypes) that comprise or are linked to a geneticmarker. Herein, analysis methods are known in the art and include, butare not limited to, PCR-based detection methods (for example, TaqManassays), microarray methods, and nucleic acid sequencing methods. Thegenes, alleles, QTL, or haplotypes to be selected for can be identifiedusing newer techniques of molecular biology with modifications ofclassical breeding strategies.

Any seed can be utilized in a method or device of the present invention.In a particular embodiment, the seed is selected from the groupconsisting of alfalfa seed, apple seed, banana seed, barley seed, beanseed, broccoli seed, castorbean seed, citrus seed, clover seed, coconutseed, coffee seed, maize seed, cotton seed, cucumber seed, Douglas firseed, Eucalyptus seed, Loblolly pine seed, linseed seed, melon seed, oatseed, olive seed, palm seed, pea seed, peanut seed, pepper seed, poplarseed, Radiata pine seed, rapeseed seed, rice seed, rye seed, sorghumseed, Southern pine seed, soybean seed, strawberry seed, sugarbeet seed,sugarcane seed, sunflower seed, sweetgum seed, tea seed, tobacco seed,tomato seed, turf seed, wheat seed, and Arabidopsis thaliana seed. In amore particular embodiment, the seed is selected from the groupconsisting of cotton seed, cucumber seed, maize seed, melon seed,soybean seed, rapeseed seed, rice seed and wheat seed. In an even moreparticular embodiment, the seed is a maize seed or a soybean seed.

In another embodiment, crops analyzed by the methods described hereininclude forage crops, oilseed crops, grain crops, fruit crops,ornamental plants, vegetable crops, fiber crops, spice crops, nut crops,turf crops, sugar crops, beverage crops, tuber crops, root crops, andforest crops.

In one embodiment, the seed is selected based on the presence or absenceof one or more characteristics that are genetically linked with a QTL.Examples of QTLs which are often of interest include but are not limitedto herbicide tolerance, disease resistance, insect or pest resistance,altered fatty acid, protein or carbohydrate metabolism, increased grainyield, increased oil, increased nutritional content, increased growthrates, enhanced stress tolerance, preferred maturity, enhancedorganoleptic properties, altered morphological characteristics, otheragronomic traits, traits for industrial uses, or traits for improvedconsumer appeal, or a combination of traits as a multiple trait index.Alternatively, the seed can be selected based on the presence or absenceof one or more characteristics that are genetically linked with ahaplotype associated with a QTL. Examples of such QTL may again includewithout limitation herbicide tolerance, disease resistance, insect orpest resistance, altered fatty acid, protein or carbohydrate metabolism,increased grain yield, increased oil, increased nutritional content,increased growth rates, enhanced stress tolerance, preferred maturity,enhanced organoleptic properties, altered morphological characteristics,other agronomic traits, traits for industrial uses, or traits forimproved consumer appeal, or a combination of traits as a multiple traitindex.

Selection of a breeding population could be initiated as early as the F₂breeding level, if homozygous inbred parents are used in the initialbreeding cross. An F₁ generation could also be sampled and advanced ifone or more of the parents of the cross are heterozygous for the allelesor markers of interest. The breeder may analyze an F₂ population toretrieve the marker genotype of every individual in the population.Initial population sizes, limited only by the number of available seedsfor analysis, can be adjusted to meet the desired probability ofsuccessfully identifying the desired number of individuals. See Sedcole,J. R. “Number of plants necessary to recover a trait.” Crop Sci.17:667-68 (1977). Accordingly, the probability of finding the desiredgenotype, the initial population size, and the targeted resultingpopulation size can be modified for various breeding methodologies andinbreeding level of the sampled population.

The selected seeds may be bulked or kept separate depending on thebreeding methodology and target. For example, when a breeder isanalyzing an F₂ population for disease resistance, all individuals withthe desired genotype may be bulked and planted in the breeding nursery.Conversely, if multiple QTL with varying effects for a trait such asgrain yield are being selected from a given population, the breeder maykeep individual identity preserved, going to the field to differentiateindividuals with various combinations of the target QTL.

Several methods of preserving single seed identity can be used whiletransferring seed from the sampling location to the field. Methodsinclude, but are not limited to, transferring selected individuals toseed tape, a cassette tray, or indexing tray, transplanting with peatpots, and hand-planting from individual seed packets.

Multiple cycles of selection can be utilized depending on breedingtargets and genetic complexity.

Advantages of using the methods of this invention include, withoutlimitation, reduction of labor and field resources required perpopulation or breeding line, increased capacity to evaluate a largernumber of breeding populations per field unit, and increased capacity toanalyze breeding populations for desired traits prior to planting. Fieldresources per population are reduced by limiting the field spacerequired to advance the desired genotypes. For example, a population of1,000 individuals may be planted at 25 seeds per row consuming a totalof 40 rows in the field. Using conventional tissue sampling, all 1,000plants would be tagged and manually sampled by scoring leaf tissue.Molecular marker results would be needed prior to pollination and onlythose plants containing the desired genetic composition would bepollinated. Thus, if it was determined that 50 seeds contained thedesired genetic composition, conventional breeding methodology wouldhave required the planting of 1000 plants to retain the desired 50seeds. By contrast, the methods of this invention allow the breeder toanalyze the 1,000 seeds in the lab and select the 50 desired seeds priorto planting. The 50 individuals can then be planted in the field,consuming only two 25 seed rows. Additionally, the methods of thisinvention do not require tagging or sampling in the field, therebysignificantly reducing the required manual labor resources.

In addition to reducing the number of field rows per population, themethods of this invention may further increase the number of populationsthe breeder can evaluate in a given breeding nursery. Using the aboveexample wherein 50 seeds out of each population of 1000 seeds containedthe desired genetic composition, a breeder applying the methods of thisinvention could evaluate 20 populations of 50 seeds each using the samefield area consumed by a single population using conventional fieldtissue sampling techniques. Even if the populations are selected for asingle allele, using a 1:2:1 expected segregation ratio for an F₂population, the breeder could evaluate 4 populations in the same fieldarea as a single field tissue sampled population.

A potential further advantage to the methods of the present invention isthe mitigation of risks associated with growing plants in certaingeographies where plants may grow poorly or experience poorenvironmental conditions, or may even be destroyed during storms. Forexample, seeds with the “best” genotype or marker composition could beplanted in geography 1 and seeds with the “next best” genotype could beplanted in geography 2. In this case geography 2 would be a backup incase any problem befell the plants grown in geography 1. This is verydifficult to do with the traditional method of taking tissue samplesfrom germinated plants for genotyping, because these plants would thenneed to be uprooted and transplanted to the second geography. Using themethods of this invention avoids the problem of transplantation and alsosimplifies the logistics of the breeding program.

The methods of the invention may further be used in a breeding programfor introgressing a trait into a plant. Such methods comprise removing asample comprising cells with nucleic acids from seeds in a population,analyzing the nucleic acids extracted from each seed for the presence orabsence of at least one genetic marker, selecting seeds from thepopulation based upon the results of the nucleic acids analysis;cultivating a fertile plant from the seed; and utilizing the fertileplant as either a female parent or male parent in a cross with anotherplant.

Examples of genetic analyses to select seeds for trait integrationinclude, without limitation, identification of high recurrent parentallele frequencies, tracking of transgenes of interest or screening forthe absence of unwanted transgenes, selection of hybrid testing seed,selection of seed expressing a gene of interest, selection of seedexpressing a heritable phenotype, identification of seed with selectedgenetic loci, and zygosity testing.

The identification of high recurrent pair allele frequencies via themethods of the present invention again allows for a reduced number ofrows per population and an increased number of populations, or inbredlines, to be planted in a given field unit. Thus, the methods of thepresent invention may also effectively reduce the resources required tocomplete the conversion of inbred lines.

The methods of the present invention further provide quality assurance(QA) and quality control (QC) by assuring that regulated or unwantedtransgenes, undesirable genetic traits, or undesirable inheritedphenotypes are identified and discarded prior to planting. Thisapplication in a QA capacity could effectively eliminate unintentionalrelease infractions. A further extension of the method is to screen forthe presence of infectious agents and remove contaminated seed prior toshipping.

The methods of the present invention may be further applied to identifyhybrid seed for transgene testing. For example, in a conversion of aninbred line at the BCnF₁ stage, a breeder could effectively create ahybrid seed lot (barring gamete selection) that was 50% hemizygous forthe trait of interest and 50% homozygous for the lack of the trait inorder to generate hybrid seed for testing. The breeder could thenanalyze all F₁ seeds produced in the test cross and identify and selectthose seeds that were hemizygous. Such method is advantageous in thatinferences from the hybrid trials would represent commercial hybridgenetics with regard to trait zygosity.

Other applications of the methods of this invention for identifying,tracking, and stacking traits of interest carry the same advantagesidentified above with respect to required field and labor resources.Generally, transgenic conversion programs are executed in multi-seasonlocations which carry a much higher land and management cost structure.As such, the impact of either reducing the row needs per population orincreasing the number of populations within a given field unit aresignificantly more dramatic on a cost basis versus temperateapplications.

The methods of this invention may be used for seeds from plants with twoor more transgenes, wherein accumulating or stacking of transgenicregions into plants or lines is achieved by addition of transgenes bytransformation, or by crossing parent plants or lines containingdifferent transgenic regions, or any combination of these. Analyses canbe conducted to select individual seeds on the basis of the presence ofone or more characteristics associated with at least one transgene. Suchcharacteristics include, but are not limited to, a transgene per se, agenetic marker linked to a transgene, mRNA expressed from a transgene,and a protein product of a transgene.

Still further, the methods of this invention may be used to improve theefficiency of the doubled haploid program through selection of desiredgenotypes at the haploid stage and identification of ploidy level toeliminate non-haploid seeds from being processed and advancing to thefield. Both applications again result in the reduction of fieldresources per population and the capability to evaluate a larger numberof populations within a given field unit.

Doubled haploid (DH) plants provide an invaluable tool to plantbreeders, particularly for generating inbred lines. A great deal of timeis spared as homozygous lines are essentially instantly generated,negating the need for multigenerational conventional inbreeding.

In particular, because DH plants are entirely homozygous, they are veryamenable to quantitative genetics studies. Both additive variance andadditive x additive genetic variances can be estimated from DHpopulations. Other applications include identification of epistasis andlinkage effects. For breeders, DH populations have been particularlyuseful in QTL mapping, cytoplasmic conversions, and trait introgression.Moreover, there is value in testing and evaluating homozygous lines forplant breeding programs. All of the genetic variance is among progeny ina breeding cross, which improves selection gain.

However, it is well known in the art that DH production process isinefficient and can be quite labor-intensive. While doubled haploidplants can occur spontaneously in nature, this is extremely rare. Mostresearch and breeding applications rely on artificial methods of DHproduction. The initial step involves the haploidization of the plantwhich results in the production of a population comprising haploid seed.Non-homozygous lines are crossed with an inducer parent, resulting inthe production of haploid seed. Seed that has a haploid embryo, butnormal triploid endosperm, advances to the second stage. That is,haploid seed and plants are any plant with a haploid embryo, independentof the ploidy level of the endosperm.

After selecting haploid seeds from the population, the selected seedsundergo chromosome doubling to produce doubled haploid seeds. Aspontaneous chromosome doubling in a cell lineage will lead to normalgamete production or the production of unreduced gametes from haploidcell lineages. Application of a chemical compound, such as colchicine,can be used to increase the rate of diploidization. Colchicine binds totubulin and prevents its polymerization into microtubules, thusarresting mitosis at metaphase, can be used to increase the rate ofdiploidization, i.e. doubling of the chromosome number These chimericplants are self-pollinated to produce diploid (doubled haploid) seed.This DH seed is cultivated and subsequently evaluated and used in hybridtestcross production.

However, processes for producing DH seed generally suffer from lowefficacy even though methods have been developed in an attempt toincrease DH production frequency, including treatment with colchicines.Outstanding issues include low production of haploid seed, reducedgamete viability resulting in diminished self-pollination for DH plantgeneration, and inadequate DH seed yield for breeding applications.

The methods of the present invention represent an advance in breedingapplications by facilitating the potential for selection at the haploidas well as the diploid seed stage. For example, in one embodiment, theinvention provides for the high-throughput analysis of a population ofhaploid seed. The method generally comprises non-destructively removinga sample from a plurality of seeds in the population and analyzing thesample for the presence of one or more characteristics indicative of atleast one genetic or chemical trait as described herein.

In another embodiment, the invention provides for the high-throughputbulking of a population of doubled haploid seeds. The method comprisesselecting one or more individual seeds exhibiting at least one preferredcharacteristic from a population of haploid seeds and producing apopulation of doubled haploid seeds from the selected seeds. Eachdoubled haploid seed is then non-destructively sampled and the samplesare analyzed for the presence or absence of one or more characteristicsindicative of at least one genetic or chemical trait. Based on theresults of the analysis, one or more individual doubled haploid seedsare selected and plants or plant tissue is cultivated from the selecteddoubled haploid seeds.

In various embodiments, the methods of the invention include analyzingseed for one or more characteristics, such as genetic markers, todetermine whether the seed is in a haploid or diploid state. The presentinvention also provides a methods for analyzing haploid and doubledhaploid seed for one or more characteristics, such as transgenes ormarkers linked to or diagnostic of transgenes, for characteristicsrelated to event performance, event evaluation, and trait integration.Further, the present invention provides a method to assay haploid seedin order to select preferred genotypic and phenotypic classes to undergodoubling.

In another embodiment, the present invention provides a basis fordetermination of linkage phase. By using seed endosperm tissue derivedfrom a diploid plant, the parental marker haplotypes can be determinedusing a genotyping system that enables detection of different allelefrequencies in DNA samples. Since endosperm tissue is triploid, with twocopies derived from the female gamete, the linkage phase of the parentalline can be derived by dissecting heterozygous progeny genotypes (seeFIG. 1). The DNA sample from endosperm tissue allows for a determinationof the ploidy level of the genetic marker. A diploid ploidy level in thegenetic marker indicates maternal inheritance and a haploid ploidy levelin the genetic marker indicates paternal inheritance.

Further, differential allele frequency data can be used to infer thegenetic linkage map but, unlike methods requiring haploid material(Gasbarra et al. 2006 Genetics 172:1325-1335), using the above-describedallele frequency calling. Determination of the genetic linkage map hastremendous utility in the context of haplotype characterization, mappingof marker (or haplotype)—trait associations. This method is particularlyrobust on a single, vs. bulked, seed basis and is thus well-suited tothe present invention.

In a particular embodiment, the invention further provides an assay forpredicting embryo zygosity for a particular gene of interest (GOI). Theassay predicts embryo zygosity based on the ratio of the relative copynumbers of a GOI and of an internal control (IC) gene per cell or pergenome. Generally, this assay uses an IC gene that is of known zygosity,e.g., homozygous at the locus (two IC copies per diploid cell), fornormalizing measurement of the GOI. The ratio of the relative copynumbers of the IC to the GOI predicts the GOI copy number in the cell.In a homozygous cell, for any given gene (or unique genetic sequence),the gene copy number is equal to the cell's ploidy level since thesequence is present at the same locus in all homologous chromosomes.When a cell is heterozygous for a particular gene (or hemizygous in thecase of a transgene), the gene copy number will be lower than the cell'sploidy level. If the GOI is not detected, the cell is null for thelocus, as can happen for a negative segregant of a transgenic event orin a mutagenized population. The zygosity of a cell at any locus canthus be determined by the gene copy number in the cell.

In another particular embodiment, the invention provides an assay forpredicting corn embryo zygosity. In corn seed, the endosperm tissue istriploid, whereas the embryo tissue is diploid. Endosperm copy number isreflective of the zygosity of the embryo: a homozygous (positive ornegative) endosperm accompanies a homozygous embryo, heterozygousendosperm (whether a GOI copy number of 1 or 2) reflects a heterozygous(GOI copy number of 1) embryo. Endosperm that is homozygous for the ICwill contain three IC copies. Endosperm GOI copy number can range from 0(homozygous negative embryo) to 3 (homozygous positive embryo); andendosperm GOI copy number of 1 or 2 is found in seed where the embryo isheterozygous for the GOI (or hemizygous for the GOI if the GOI is atransgene). The endosperm GOI copy number (which can range from 0 to 3copies) can be determined from the ratio of endosperm IC copy number toendosperm GOI copy number (which can range from 0/3 to 3/3, that is,from 0 to 1), which can then be used to predict zygosity of the embryo.

Copy numbers of the GOI or of the IC can be determined by any convenientassay technique for quantification of copy numbers, as is known in theart. Examples of suitable assays include, but are not limited to, RealTime (TaqMan®) PCR (Applied Biosystems, Foster City, Calif.) andInvader® (Third Wave Technologies, Madison, Wis.) assays. Preferably,such assays are developed in such a way that the amplificationefficiency of both the IC and GOI sequences are equal or very similar.For example, in a Real Time TaqMan® PCR assay, the signal from asingle-copy GOI (the source cell is determined to be heterozygous forthe GOI) will be detected one amplification cycle later than the signalfrom a two-copy IC, because the amount of the GOI is half that of theIC. For the same heterozygous sample, an Invader® assay would measure aGOI/IC ratio of about 1:2 or 0.5. For a sample that is homozygous forboth the GOI and the IC, the GOI signal would be detected at the sametime as the IC signal (TaqMan®), and the Invader assay would measure aGOI/IC ratio of about 2:2 or 1.

These guidelines apply to any polyploid cell, or to haploid cells (suchas pollen cells), since the copy number of the GOI or of the IC remainproportional to the genome copy number (or ploidy level) of the cell.Thus, these zygosity assays can be performed on triploid tissues such ascorn endosperm. Furthermore, the copy number for a GOI can be measuredbeyond 2 copies or at numerically different values than the ploidy ofthe cell. The method is still appropriate for detecting GOI inpolyploids, in some transgenic events with >2 copies of the insertedtransgene, after replication of the GOI by transposition, when the GOIexists on autonomously replicating chromosomes or plasmids and othersituations.

In plant breeding, it is useful to determine zygosity at one or moreloci for the purpose of evaluating the level of inbreeding (that is, thedegree of gene fixation), segregation distortion (i.e., in transgenicgermplasm, maternal inheritance testing or for loci that affect thefitness of gametes), and the level of outbreeding (i.e., the relativeproportion of homozygosity and heterozygosity). Similarly, the extent ofzygosity at one or more loci can be used to estimate hybridity andwhether a particular seed lot meets a commercial or regulatory standardfor sale as certified hybrid seed. In addition, in transgenic germplasm,it is useful to know the ploidy, or copy number, in order to distinguishbetween quality events and to aid in trait integration strategies.

In another embodiment, the present invention provides a basis forimproving the ability to monitor one or more germplasm pools for shiftsin the frequencies of one or more genetic characteristics, wherein saidgenetic characteristics include markers, alleles, and haplotypes.Methodology is known in the art to compare genetic marker frequencybetween recently derived populations and their ancestral lines in orderto identify those genetic loci that are increasing in frequency overtime (U.S. Pat. Nos. 5,437,697 and 5,746,023). Those loci withfrequencies that exceed the expected allele frequency are inferred tohave been subject to selection. Further, given that the predominantselection criterion in breeding programs is yield, it is expected thatthose increasingly frequent alleles may be linked to yield.

In a particular embodiment, the present invention provides a method toenable haplotype-assisted breeding. By comparing the frequency ofhaplotypes in emerging elite lines with the haplotype frequency in theancestral elite lines (as determined via pedigree analysis),identification of haplotypes that are deviating from the expectedhaplotype frequency is possible. Further, by evaluation of haplotypeeffect estimates for said haplotypes, it is also possible to link saidhaplotypes of increasing frequency with phenotypic outcomes for a suiteof agronomic traits. The haplotype composition of individual seedssampled from a plurality of seeds can be determined using geneticmarkers and the seeds with preferred haplotypes are selected andadvanced. Thus, more informed breeding decisions and establishment ofsuperior line development programs is enabled by this technology.

EXAMPLES

The following examples are merely illustrative, and not limiting to thisdisclosure in any way.

Example 1

This example describes an assay for predicting the zygosity of cornembryos using an internal control (IC) gene homozygous at the locus(i.e., two IC copies in the diploid embryo and three IC copies in thetriploid endosperm). In an inbred line of a diploid (or higher ploidy)organism such as corn, the endogenous internal control is typicallyhomozygous; transgenic events in such organisms at the first generation(termed “R0” in corn) are typically hemizygous (that is, the transgeneis typically present in only one of the two or more homologouschromosomes). Corn (Zea mays) is a diploid organism, thus a “singlecopy” R0 event has one copy of the GOI per cell, but 0.5 copies perhaploid genome, a “two copy” R0 event has two copies of the GOI percell, but 1 copy per haploid genome, and so forth.

In this example, tubulin was used as the IC gene, and the GOI was atransgene encoding neomycin phosphotransferase II (NPT II), which isused for kanamycin resistance selection. Endosperm (triploid) tissue wastaken from seed (either by hand sampling or by scraping a seed with anautomated sampler of the present invention). The endosperm-sampled seedwas germinated, and leaf tissue (diploid) from successfully germinatedplants was also sampled for genetic analysis. The leaf tissue correlatesdirectly with embryo zygosity and was thus used to demonstrate thatendosperm zygosity generally predicted zygosity of the embryo and toconfirm homozygosity calls from the endosperm. Total genomic DNA wasextracted from endosperm tissue and from leaf tissue, and quantitativelyanalyzed using an Invader® assay with oligonucleotide probes specificfor the gene of interest, NPT II, or for the internal control gene,tubulin. The ratio of the GOI to IC was measured using conventionalmolecular biology techniques. See Table 1. A summary of results ofmultiple experiments are shown in Table 2.

Results indicated that endosperm zygosity generally predicted zygosityof the embryo (as indicated by the leaf zygosity) and was reliable inpredicting homozygosity for all seeds that germinated. Furthermore,endosperm zygosity analysis gave few false-negative homozygouspredictions (especially when the endosperm tissue was obtained with theautomated sampler). These results demonstrate that for a cell of a knownploidy level, the ratio of copy number of a GOI to that of an ICindicates the zygosity of that cell. Furthermore, the zygosity assay ofthe present invention can predict zygosity of one tissue based on thezygosity of another, that is, the assay can predict the embryo zygositybased on the endosperm zygosity.

TABLE 1 Automated Manual Ratio Automated Zygosity Ratio Manual Zygosity1.39 Heterozygous 1.42 Heterozygous 0.14 neg homozygous 0.12 neghomozygous 0.08 neg homozygous 0.08 neg homozygous 0.13 neg homozygous0.10 neg homozygous 0.10 neg homozygous 0.08 neg homozygous 1.55Heterozygous 1.38 Heterozygous 0.84 Heterozygous 1.45 Heterozygous 0.14neg homozygous 1.48 Heterozygous 1.48 Heterozygous 1.37 Heterozygous1.39 Heterozygous 1.47 Heterozygous 2.03 POS homozygous 1.93 POShomozygous 0.13 neg homozygous 0.05 neg homozygous 1.71 Inconclusive1.81 POS homozygous 0.81 Heterozygous 1.41 Heterozygous 1.84 POShomozygous 1.77 POS homozygous 1.54 Heterozygous 1.43 Heterozygous 1.48Heterozygous 1.50 Heterozygous 0.92 Heterozygous 1.40 Heterozygous 1.51Heterozygous 1.42 Heterozygous 1.60 Heterozygous 1.37 Heterozygous 0.86Heterozygous 1.47 Heterozygous 1.81 POS homozygous 2.02 POS homozygous0.15 neg homozygous Low DNA 1.89 POS homozygous 1.85 POS homozygous 0.21neg homozygous 0.10 neg homozygous 0.09 neg homozygous 0.11 neghomozygous 0.89 Heterozygous 1.50 Heterozygous 1.50 Heterozygous 1.37Heterozygous 1.82 Inconclusive 2.02 POS homozygous 2.14 POS homozygous0.99 inconclusive 1.22 Heterozygous 1.44 Heterozygous 2.22 POShomozygous 2.24 POS homozygous 0.79 Heterozygous 1.40 Heterozygous 1.23Heterozygous 1.47 Heterozygous 1.49 Heterozygous 1.38 Heterozygous 1.33Heterozygous 1.37 Heterozygous

TABLE 2 Number of Number of Number of Number of homozygous predictedconfirmed false negative seeds homozygous homozygous homozygousEndosperm identified by seeds that calls based calls based on samplingendosperm did not on leaf endosperm method analysis germinate analysisanalysis Hand 8 out of 36 0 8 (all) 5 (13.9%) Automated 6 out of 24 1 50 Hand 6 out of 36 0 6 (all) 2 (5.6%) Automated 6 out of 24 1 5 0 Hand 5out of 36 0 5 (all) 7 (19.4%) Automated 7 out of 24 2 5 0 Hand 7 out of36 1 6 0 Automated 5 out of 24 2 3 0

Example 2

This example demonstrates the use of the methods of the presentinvention in a program for marker-assisted selection of soybeans for LowLinolenic Acid.

Soybean is the most valuable legume crop, with many nutritional andindustrial uses due to its unique chemical composition. Soybean seedsare an important source of vegetable oil, which is used in food productsthroughout the world. The relatively high level (usually about 8%) oflinolenic acid (18:3) in soybean oil reduces its stability and flavor.Hydrogenation of soybean oil is used to lower the level of linolenicacid (18:3) and improve both stability and flavor of soybean oils.However, hydrogenation results in the production of trans fatty acids,which increases the risk for coronary heart disease when consumed. Thedevelopment of low linolenic acid soybean has been complicated by thequantitative nature of the trait. The low linolenic acid soybeanvarieties that have been developed have been found to yield poorly,limiting their usefulness in most commercial settings. Developing aproduct with commercially significance seed yield is a high priority inmost soybean cultivar development programs.

An example of the application of the methods of the present invention isselection of soybean plants with both high yield and decreased linolenicacid content. Soybean progeny performance as it relates to low linolenicacid relies mainly on two major quantitative trait locus (QTL) atFad3-1b and Fad3-1 c. Analysis of segregating plants demonstrated thatFad3-1b and Fad3-1c additively control linolenic content in soybean.Therefore, by using a combination of markers for Fad3-1b and Fad3-1c, abreeder using the invention can accurately predict linolenic acidcontent in soybean plants. The markers can be used to infer thegenotypic state of a seed at any stage in the breeding process, forexample, at the finished inbred line stage, or the F₁, F₂, F₃, etc.

A seminal F₁ hybrid can be produced by crossing two inbred soybean lines(for example, crossing a plant containing the Fad3-1b and/or Fad3-1calleles associated with decreased linolenic acid content to a plantlacking these alleles) followed by natural self-pollination. Since themarkers can be used to infer the genotypic state of a single seedobtained from an intermating of such inbred lines, early generation(i.e., F₂) marker-assisted breeding can be conducted.

Soybean seed at ambient temperature and humidity typically equilibrateto 8% moisture on a dry weight basis. Soybean seed at this level ofmoisture tends to split when sampled. To reduce splitting, seed shouldbe humidified to moisture level of 12%. When pretreated in this manner,splitting is significantly reduced to <5%.

The selected F₂ seed that have the desired genotype may be bulked orkept separate depending on the breeding objectives. If multiple QTL withvarying effects were being selected from a given population, the breedercould preserve single seed identity to differentiate individuals withvarious combinations of the target resistance QTL. These seeds could beplanted in the field with appropriate field identification. Severalmethods of preserving single seed identity can be used whiletransferring seed from the sampling lab to the field. Methods includetransferring selected individuals to horticultural seed tape that couldalso include radio frequency identification to aid in the identificationof the individual genotyped seed. Other methods would be to use anindexing tray, plant seeds in peat pots and then transplant them, orhand plant from individual seed packets.

Example 3

This example demonstrates the use of the methods of the presentinvention in a program for recurrent parent alleles in a backcrossbreeding program.

The methods of the present invention can be used for selection oftransgenes as well as identification of recurrent parent alleles. Theidentification of genotypes with desired recurrent parent allelefrequencies before planting allows the number of rows per population tobe reduced throughout the entire breeding program along with an increasein the number of populations included in the conversion program within agiven field unit. This results in improved land usage, reduced land andlabor costs, etc.

An example of analyzing endosperm tissue from corn for recurrent parentalleles in a backcross breeding program is shown in FIG. 1.

Example 4

This example demonstrates the use of the methods of the presentinvention for use in DNA line fingerprinting and linkage phasedetermination.

Combined with bulking of a single seed's DNA, line fingerprinting couldbe accomplished without the need to sample the line in the field.

By using seed endosperm tissue (seed coat in soybean) derived from adiploid plant, the parental marker haplotypes can be determined using agenotyping system that enables detection of different allele frequenciesin DNA samples. Since endosperm tissue is triploid, with two copiesderived from the female gamete, the linkage phase of the parental linecan be derived by dissecting heterozygous progeny genotypes. The DNAsample from endosperm tissue allows for a determination of the ploidylevel of the genetic marker. A diploid ploidy level in the geneticmarker indicates maternal inheritance and a haploid ploidy level in thegenetic marker indicates paternal inheritance.

Example 5

This example demonstrates the methods of the present invention forevaluating transgenic seed for segregation distortion. Seeds of an F1cross between Line A (Homozygous Event 1 and Event 2) and Line B(Homozygous Event 1) were induced in a maternal haploid inductionisolation. The resulting kernels were selected using plumule color toobtain a population of putative haploid seed.

Individual putative haploid kernels from the population of putativehaploid seed were selected and non-destructively sampled using anautomated seed sampler system as generally described in U.S. patentapplication Ser. No. 11/213,435 (Publication No. US 2006/004624), whichis hereby incorporated by reference in its entirety. Markers wereapplied to the samples to determine the presence of the Event 2 gene andthe Event 1 gene. The sampling process clips off some pericarp andendosperm tissue and uses this as the base for analysis. It is importantto note that endosperm tissue is triploid and contains geneticcontribution from both parents. If the gene of interest is detectedusing this method, it accurately predicts the presence of the desiredgene in the haploid embryo. For the purposes of this study, samples from180 kernels were analyzed and data were obtained on 175 due to samplingissues.

As shown in Table 3 below, each of the seed samples tested positive forthe Event 1 gene as expected and approximately 50% of the seed samplestested positive for the Event 2 gene, confirming no segregationdistortion.

TABLE 3 Event 2 Event 1 Pedigree Chromosome  6  8 Position  38  63Parental Checks Line A Pos Pos Line B Neg Pos KHI1 Neg Neg SelectedKernels 175 175 Total Positive 92/175 175/175 Total Negative 83/175 0/175

Results of this study indicate that individual gene traits can beselected on a haploid basis using high throughput, nondestructive seedsampling as a screening mechanism.

Example 6

This example demonstrates the utility of automated, high-throughputsampling in the preselection of haploid seed from a population of seeds.

The experiment comprised sampling 20 F2 populations using anondestructive, high throughput seed sampling system and analyzing thesamples to verify the pre-selection of haploid seed. Each population ofF2 seed was nondestructively sampled or the F2 plants were tissuesampled for DNA analysis. The nondestructive seed samples were collectedfrom individual seeds in the population of seeds using an automated seedsampler system as generally described in U.S. patent application Ser.No. 11/213,435 (Publication No. US 2006/004624), which is herebyincorporated by reference in its entirety. Selection of desirablegenotypes was based on selecting materials with the greatest allelicfrequencies of the desired haplotypes based on modeling parameters. Theselected F2 plants were pollinated with haploid inducing malepollinators and the resulting seed is harvested. Following harvest,haploid kernels were sorted out from the non-haploid seed and thehaploids were sampled on a kernel basis using nondestructive, highthroughput sampling and subsequent genotyping. The preferred haploidseed was selected and subjected to a chromosome doubling procedure toproduce doubled haploids. This approach allows non-preferred genotypesto be culled before doubling and increases the frequency of preferredmaterial that is processed through the resource intensive doublingprocess.

Results comparing the selected haploid seed and illustrating theefficacy of this approach are shown in FIG. 2.

When introducing elements or features of embodiments herein, thearticles “a”, “an”, “the” and “said” are intended to mean that there areone or more of such elements or features. The terms “comprising”,“including” and “having” are intended to be inclusive and mean thatthere may be additional elements or features other than thosespecifically noted. It is further to be understood that the methodsteps, processes, and operations described herein are not to beconstrued as necessarily requiring their performance in the particularorder discussed or illustrated, unless specifically identified as anorder of performance. It is also to be understood that additional oralternative steps may be employed.

The description of the disclosure is merely exemplary in nature and,thus, variations that do not depart from the gist of the disclosure areintended to be within the scope of the disclosure. Such variations arenot to be regarded as a departure from the spirit and scope of thedisclosure.

What is claimed is:
 1. A method of evaluating seeds for a desired trait,the method comprising: removing tissue samples from individual seedsusing an automated seed sampler without affecting germination viabilityof the seeds; analyzing the tissue samples for one or morecharacteristics indicative of a desired trait; and selecting the sampledseeds that include the desired trait.
 2. The method of claim 1, whereinanalyzing the tissue samples for one or more characteristics indicativeof a desired trait includes analyzing the tissue samples for the one ormore characteristics indicative of the presence of the desired trait. 3.The method of claim 1, wherein analyzing the tissue samples for one ormore characteristics indicative of a desired trait includes analyzingthe tissue samples for the one or more characteristics indicative of theabsence of the desired trait.
 4. The method of claim 1, wherein thedesired trait is selected from the group consisting of a genetic marker,a single nucleotide polymorphism, a simple sequence repeat, arestriction fragment length polymorphism, a haplotype, a tag SNP, analleles of a genetic marker, a gene, a DNA-derived sequence, anRNA-derived sequence, a promoter, a 5′ untranslated region of a gene, a3′ untranslated region of a gene, microRNA, siRNA, a QTL, a satellitemarker, a transgene, mRNA, ds mRNA, a transcriptional profile, and amethylation pattern.
 5. The method of claim 1, wherein the desired traitis genetically linked with a haplotype associated with a QTL selectedfrom the group consisting of herbicide tolerance, disease resistance,insect or pest resistance, altered fatty acid, protein or carbohydratemetabolism, increased grain yield, increased oil, increased nutritionalcontent, increased growth rates, enhanced stress tolerance, preferredmaturity, enhanced organoleptic properties, altered morphologicalcharacteristics, other agronomic traits, traits for industrial uses,traits for improved consumer appeal, and a combination of traits as amultiple trait index.
 6. The method of claim 1, wherein the desiredtrait is genetically linked with a QTL selected from the groupconsisting of herbicide tolerance, disease resistance, insect or pestresistance, altered fatty acid, protein or carbohydrate metabolism,increased grain yield, increased oil, increased nutritional content,increased growth rates, enhanced stress tolerance, preferred maturity,enhanced organoleptic properties, altered morphological characteristics,other agronomic traits, traits for industrial uses, traits for improvedconsumer appeal, and a combination of traits as a multiple trait index.7. The method of claim 1, wherein removing tissue samples fromindividual seeds using an automated seed sampler without affectinggermination viability of the seeds includes removing the tissue samplesfrom the individual seeds using the automated seed sampler whilemaintaining the germination viability of greater than fifty percent ofthe seeds.
 8. The method of claim 1, wherein selecting the sampled seedsthat include the desired trait includes selecting the sampled seeds forbreeding.
 9. The method of claim 8, wherein the individual seeds are F₁generation seeds.
 10. The method of claim 8, wherein the individualseeds are F₂ generation seeds.
 11. The method of claim 1, furthercomprising using the selected seeds in a breeding program to introgressthe desired trait into a plant.
 12. The method of claim 1, wherein theindividual seeds are from one or more transgenic plants.
 13. The methodof claim 1, further comprising bulking the selected seeds.
 14. Themethod of claim 1, further comprising preserving single seed identity ofthe selected seeds.
 15. The method of claim 1, further comprisingcultivating plants from the selected seeds.
 16. The method of claim 15,further comprising: collecting seeds from the cultivated plants;removing tissue samples from the collected seeds using the automatedseed sampler without affecting germination viability of the seeds;analyzing the tissue samples for one or more characteristics indicativeof the desired trait; and selecting the seeds that include the desiredtrait, marker, and/or genotype.
 17. The method of claim 1, wherein theone or more characteristics are selected from the group consisting ofproteins, oils, carbohydrates, fatty acids, amino acids, biopolymers,pharmaceuticals, starch, fermentable starch, secondary compounds, andmetabolites.
 18. The method of claim 1, wherein removing tissue samplesfrom individual seeds using an automated seed sampler without affectinggermination viability of the seeds includes removing the tissue samplesfrom endosperm portions of the seeds.
 19. The method of claim 1, furthercomprising coating the selected seeds with a polymer and/or a fungicideafter removing the tissue samples therefrom to further preservegermination viability.